This application is a 371 of PCT/US95/15098, filed Nov. 27, 1995.
1. Field of the Invention
The present invention is generally in the field of mammalian gene expression systems used for the cell culture production of proteins.
In particular, the present invention concerns new gene expression systems for increasing the efficiency and the amount of protein production in eukaryotic cell lines and, in particular, the well-known chinese hamster ovary (CHO) cell culture systems which utilize the dihydrofolate reductase (DHFR)xe2x80x94methotrexate (MTX) gene expression amplification system for the production of a wide variety of medically and veterinarily important proteins on a commercial scale. These new expression systems which employ new gene-expression enhancing vectors containing an anti-sense DHFR gene sequence, permit higher levels of protein production in eukaryotic cells at significantly lower levels of MTX, i.e. these eukaryotic cells are rendered more MTX-sensitive.
2. Description of the Background Art
DHFR/TX Mediated Gene Amplification
Many medically and veterinarily important proteins are produced using the CHO cell culture system in which gene expression is amplified by the DHFR/MTX system (Kaufman, R. J., Methods in Enzymol. 185, 537-566 (1990)). This system was developed in the early 1980""s (see Axel, U.S. Pat. No. 4,399,216; Ringold et al., J. Mol. Appl. Genet. 1:165-175 (1981); Kaufman et al., J. Mol. Biol. 15:601-621 (1982)). Examples of proteins produced by this system are interleukins, interferons, receptors, human factor IX, human factor VIII, bovine luteinizing hormone, and others.
In the known CHO DHFR/MTX protein production systems, the CHO cell lines are usually DHFRxe2x88x92 and as such, they can only grow in the essential absence of methotrexate. These CHO cell lines are then transformed or co-transformed with a plasmid/vector carrying a gene sequence encoding the protein of choice to be expressed together with the DHFR gene carried either on the same vector or on a different one. When expressed, the enzyme DHFR serves as a selectable marker for these transformed cells. The vector is usually of the type which can undergo stable recombination and hence subsequent stable incorporation into the genome of CHO DHFRxe2x88x92 cells thereby rendering such cells DHFR+ in a stable and constitutive manner.
The positively transformed DHFR+ cells are selected by growing the cells in a standard culture medium, in which DHFRxe2x88x92 cells cannot grow, and subjecting the cell cultures to a number of culture cycles or passages. Such a medium usually contains sufficient methotrexate to kill DHFR cells but which is generally not lethal to the DHFR+ cells. This gives rise to stably transformed DHFR+ cells, i.e., in which the entire transforming vector or co-transforming vectors or the essential portions thereof such as the DHFR+ sequence and sequences adjacent thereto which include the gene encoding the protein of choice are stably integrated into the host chromosome. These stably transformed DHFR+ cells are further cultured and cloned, i.e., individual colonies of cells are taken and cultured separately to provide a number of cloned, transformed DHFR+ cell lines. These DHFR+ cell lines are then examined for their ability to express the desired protein and those cell lines showing good expression (i.e., expressing the protein of choice in its expected form either as an intact protein or as an intact fusion product, depending on how the gene encoding the protein of choice was originally constructed on the transforming vector) are selected.
Axel et al., U.S. Pat. No. 4,399,216, discloses a system for co-transforming eukaryotic cells with a foreign DNA encoding a desired proteinaceous material and with an unlinked DNA encoding a selectable phenotype such as DHFR conferring methotrexate resistance. Alternatively, Axel et al. discloses amplifying a gene encoding a desired protein linked to the DNA encoding a selectable phenotype by challenging with successively higher amounts of the selecting agent.
The addition of MTX during cell culture causes gene amplification of gene sequences at and around the DHFR sequence, such as large stretches of flanking DNA that include the gene sequence encoding the desired protein (when unlinked DNAs are cotransfected into a cell, they tend to form a cointegrate that link the DNAs prior to integration into the host genome by non-homologous recombination). This results in an increased number of copies of these sequences and consequently, also results in elevated levels of both DHFR and the desired protein. The degree of amplification is regulated by the MTX inhibitory effect of DHFR.
However, the above CHO DHFR/MTX system has a number of drawbacks, the major one being that the necessary constitutive expression of the DHFR gene during cell culture results in increased levels of DHFR which act to inhibit the effect of MTX. Thus, as the cell culture progresses through successive stages of amplifying stable transfectants from the previous stage, more and more MTX is required for gene amplification until a limit is reached whereby the elevated MTX levels become toxic to the cells; in other words, the upper concentration limit of MTX to which the cells are still MTX-resistant is reached. Accordingly, the current CHO protein production systems have an upper limit as to the amount of desired protein that can be produced. The level of constitutive heterologous (desired) protein expression is relatively limited; for example, only production levels of as high as 10-30 mg/l of culture can be obtained after MTX treatment. Consequently, in order to further increase the amounts of protein produced in these systems, either additional cultures are required or larger cultures need to be grown, which adds considerably to the production costs. As mentioned above, current CHO protein production systems are employed for the production of medically and veterinarily important proteins on a commercial scale. There has therefore been a long-felt need to improve these systems to increase the amount of desired protein produced, on the one hand, and on the other hand, to reduce the costs for producing this increased amount of protein.
Anti-Sense DNA
Anti-sense RNA is transcribed from an upstream promoter of a coding sequence oriented in the anti-sense direction, i.e., opposite the normal or sense direction of the DNA and its transcribed sense RNA. The expression of anti-sense RNA complementary to the sense RNA is a powerful way of regulating the biological function of RNA molecules. Through the formation of a stable duplex between the sense RNA and anti-sense RNA, the normal or sense RNA transcript can be rendered inactive and untranslatable.
In prokaryotes, anti-sense RNA is believed to control plasmid COLE1 replication (Tomizawa et al., Proc. Nat""l. Acad. Sci. USA 78:1421, 1981; Lacatena et al., Nature 294:623,1981) and regulation of outer membrane protein production (Mizuno et al., Proc. Nat""l Acad. Sci. USA 81:1966,1984) as well as many others. Izant et al., Cell 36:1007 (1984), showed that anti-sense RNA also inhibits gene expression in eukaryotes. They constructed a plasmid with a promoter directing the transcription of an anti-sense RNA complementary to the normal thymidine kinase (tk) transcript which substantially reduced expression of the normal thymidine kinase gene.
Besides the thymidine kinase gene and the outer membrane protein genes OmpF and OmpC, anti-sense DNA sequences have been used to express anti-sense RNA complementary to normal or sense RNA transcripts of numerous genes. As an example, Kaufman et al., U.S. Pat. No. 4,912,040, discloses a system for expressing an anti-sense GRP78 DNA sequence capable of hybridizing to part or all of the endogenous GRP78 (similar to immunoglobulin heavy chain binding protein)xe2x80x94encoding mRNA transcript and thereby preventing its translation into GRP78 protein.
Anti-sense DNA to DHFR or DHFR-TS complex has also been reported. Wang et al., Nucl. Acids Res. 21:4383-4391 (1993) transfected expression vectors carrying anti-sense DHFR cDNAs into a DHFR over-expressing KB (epidermoid carcinoma) cell line to quantitatively evaluate stoichiometric effects on RNA hybrid duplex formation.
Sartorius et al., Nucl. Acids Res. 19:1613 (1991) showed inhibition of the in vitro translation of Plasmodium falciparum mRNA coding for the endogenous bifunctional enzyme dihydrofolate reductase-thymidylate synthase (DHFR-TS) with oligodeoxynucleotides directed against the translation initiation site or a site in the TS-coding region. Maher et al., Nucl. Acids Res. 16:3341-3358 (1988) also used anti-sense oligonucleotides to test arrest of in vitro translation of human DHFR mRNA. However, Maher""s oligonucleotides contained either anionic diester or neutral methyl phosphonate internucleoside linkages prepared by automated synthesis.
Shotkoski et al., J. Trop. Med. Hyg. 50:433-439 (1994) expressed the mosquito DHFR gene, in both the sense and anti-sense orientations, under the control of a temperature-inducible promoter, in mosquito cells. Expression of the DHFR sense gene had little effect on cell growth, while expression of the anti-sense DHFR compromised cell growth and viability. Clones transfected with the sense construct retained significantly higher copy numbers of foreign DNA than did those receiving the anti-sense vector.
However, anti-sense DHFR has not previously been used to increase the responsiveness of DHFR+ cells to methotrexate.
The present invention is based on the surprising and unexpected discovery that upon transfection of a eukaryotic cell line that is DHFR+, MTX-resistant and capable of producing a desired protein, with a vector encoding an anti-sense DHFR sequence, the cell line became more responsive (sensitive) to MTX, thus allowing higher levels of gene amplification and hence higher production levels of the desired protein at lower MTX levels. Even higher levels of protein production can be achieved by increasing MTX levels to the upper limit where the cells are still MTX-resistant. In some cases, the increased level of protein production was 500% (5-fold) higher than that achieved for control cultures not transformed with anti-sense DHFR-encoding plasmid.
It is therefore an object of the present invention to provide an anti-sense DHFR sequence, which may be introduced into eukaryotic MTX-resistant cells and upon expression of the anti-sense DHFR sequence, lead to a reduction in the amount of DHFR produced in the cells and hence a reduction in the amount of MTX required for gene amplification, with the result that increased amounts of protein can be produced at lower MTX levels (i.e. the cells are rendered more MTX-sensitive).
The present invention also provides an expression enhancing system for regulating the amount of protein production in eukaryotic cells having protein production levels regulated by a DHFR/MTX regulatory system. The expression enhancing system comprises an expression enhancing vector according to the present invention, that is capable of transfecting eukaryotic cells which are DHFR+ and MTX-resistant, and which produce, under suitable conditions, a desired protein product and, when expressed in the eukaryotic cell line, is capable of producing anti-sense DHFR RNA which is complementary to the normal DHFR mRNA produced in the same cells. The anti-sense DHFR RNA can specifically hybridize to normal DHFR in the eukaryotic cell line and consequently, can increase both the MTX sensitivity and the desired protein production at lower MTX levels in these eukaryotic cells.
The above expression system of the invention may be used to control the expression of a desired protein selected from the group of desired proteins consisting of the known medically and veterinarily important proteins produced in CHO cells under DHFR/MTX regulation, for example, interleukins (ILs), interferons (IFNs), receptors and others.
Accordingly, the present invention provides an expression enhancing vector for regulating the expression of the normal DHFR gene carried by an eukaryotic cell line, the expression enhancing vector comprising:
a) a double stranded sequence encoding the DHFR gene or a portion thereof in the anti-sense or reverse orientation instead of the sense or normal orientation, the anti-sense DHFR sequence being transcribable by RNA polymerase under the control of a promoter sequence to yield an anti-sense RNA product that is complementary to the normal DHFR mRNA sequence or a portion thereof, and capable of specifically hybridizing to the normal DHFR mRNA sequence, sufficiently to inhibit translation of the normal DHFR mRNA;
b) a promoter sequence situated adjacent to the anti-sense DHFR sequence and controlling the expression of the anti-sense DHFR sequence. The promoter sequence is located upstream of the 5xe2x80x2 end of the anti-sense DHFR sequence so that the 5xe2x80x2xe2x86x923xe2x80x2 directionality of the promoter sequence is in phase with the 5xe2x80x2xe2x86x923xe2x80x2 directionality of the anti-sense sequence enabling transcription to proceed from the promoter in the 5xe2x80x2xe2x86x923xe2x80x2 direction to yield an anti-sense DHFR RNA product; and, optionally,
c) a genetic marker gene sequence encoding a product, the expression of which is readily screenable or selectable in cells transfected with and expressing the expression enhancing plasmid.
The present invention also provides a method for regulating the level of desired protein production in eukaryotic cells which comprises genetically manipulating an eukaryotic cell line so that it expresses a protein of interest, together with both DHFR (so that it is MTX-resistant) and an anti-sense DHFR RNA which hybridizes with normal DHFR mRNA to inhibit the translation thereof, thus leading to a decrease in DHFR production and in the level of inhibition of MTX. This decreased level of MTX-inhibition results in an increase in the amplification of the desired gene and its subsequent expression. In one embodiment this genetic manipulation involves:
a) constructing an eukaryotic cell line that is DHFR+, MTX-resistant and capable of producing a desired protein, the level of production of which is at least partly regulated by the MTX-amplifiable copy number of the gene encoding the desired protein. (The level of MTX-induced amplification is regulated by the level of normal DHFR expression in these cells); and
b) transfecting the eukaryotic cell line with an expression enhancing vector according to the present invention.
It will be appreciated, however, that the genetic elements (the gene encoding the protein of interest, the DHFR gene, and the anti-sense DHFR gene) can be introduced in any order, and on the same or different vectors, provided that the DHFR gene and the gene of interest are or become associated such that amplification of the DHFR gene results in amplification of the gene of interest.
The new expression system provides for both an increase in the amount of protein produced and a reduction in the cost of producing medically and veterinarily important proteins in CHO cell cultures.
The method of the present invention results in an increase in expression, which is to a level preferably at least about 200%, more preferably at least about 300%, most preferably at least about 500%, that achieved in control DHFR+ cultures not transferred with the anti-sense sequence.
This method is generalizable to other gene amplification systems in which a directly amplifiable gene (the counterpart of the DHFR gene in our model system) whose expression product is required to protect the cell from a toxic agent (the counterpart of the methotrexate in our model system) is cotransfected with a gene of interest, which thereby becomes indirectly amplifiable by exposing the cell to the toxic agent. This exposure causes the cell to elevate expression of the protective expression product in such a manner as to result in the elevated expression of the gene of interest. The method would then involve expression of anti-sense RNA which inhibits translation of the mRNA encoded by the directly amplifiable gene so as to render the cell more sensitive to the elicitor.
Additional aspects and embodiments of the invention are set forth or readily arise from the drawings described below, or from the following detailed description of the preferred embodiments of the invention.